Antibodies having binding specificities for at least two different antigens, called bispecific antibodies (BsAbs), have been engineered. Unlike classical antibodies which comprise two identical heterodimer (i.e. a light chain portion and a heavy chain portion) “arms” wherein each arm comprises an antigen binding site (e.g. a Fab region), bispecific antibodies have different sequences in each of the two arms (e.g. Fab regions) so that each arm of the Y-shaped molecule binds to a different antigen or different epitope of the same antigen.
By binding two different antigenic molecules or different epitopes of the same antigen, BsAbs offer a wide variety of clinical applications as targeting agents for in vitro and in vivo diagnostics and immunotherapies. Bispecific antibodies are also advantageous for in vitro or in vivo diagnoses of various disease states, including cancer. For example, one arm of the BsAb can be engineered to bind a tumor-associated antigen and the other arm to bind a detectable marker.
BsAbs can be used to direct a patient's cellular immune defense mechanisms to a tumor cell or an infectious agent (e.g. virally infected cells such as HIV or influenza virus; protozoa such as Toxoplasma gondii). In particular, one can redirect immune modulated cytotoxicity by engineering one arm of the BsAb to bind to a desired target (e.g. a tumor cell or pathogen) and the other arm of the BsAb to bind to a cytotoxic trigger molecule, such as the T-cell receptor or a Fc gamma receptor, thereby activating downstream immune effector pathways. Using this strategy, BsAbs which bind to the Fc gamma RIII have been shown to mediate tumor cell killing by natural killer (NK) cell/large granular lymphocyte (LGL) cells in vitro and to prevent tumor growth in vivo. Alternatively, targeting two separate antigens or targets related to the therapeutic indication can enhance specificity and reduce unwanted interaction, thereby widening the therapeutic index.
Although bispecific antibodies posses certain advantages over canonical bivalent monospecific classical antibodies, use of bispecific antibodies has been hindered by the expense in obtaining BsAbs in sufficient quantity and purity.
To produce multispecific proteins, e.g. bispecific antibodies and other heterodimers or heteromultimers, it is desirable to use methods that favor formation of the desired heteromultimer over homomultimer(s). One method for obtaining Fc-containing BsAbs remains the hybrid hybridoma technique, in which two antibodies are co-expressed. However, this approach is inefficient with respect to yield and purity, the desired heteromultimer often being difficult to further purify from a relatively large level of contaminant comprising improperly paired polypeptide chains.
Other techniques to favor heteromultimer formation and reduce improper matching involve engineering sterically complementary mutations in multimerization domains at the CH3 domain interface, referred to as a “knobs-into-holes” strategy as described by Ridgway et al. (U.S. Pat. No. 5,731,168) and Merchant et al. (U.S. Pat. No. 7,183,076).
Techniques that replace one or more residues that make up the CH3-CH3 interface in both CH3 domains with a charged amino acid for promoting the heterodimer formation have also been described by Strop et al. (WO2011/143545).
A recent review also discusses various approaches for overcoming chain association issues when generating bispecific antibodies (Klein et al., mAbs 4(6): 653-663 (2012)).
However, most of these techniques are directed to ensuring proper pairing of the heavy chain polypeptides and do not address the further matching of each light chain polypeptide with its corresponding heavy chain polypeptide to provide a functional antigen-binding site. Thus, production of desired bispecific antibodies remains a technically difficult and costly process not commercially feasible due to the high cost of goods.
Therefore, there is a long-felt need in the art for methods for engineering bispecific antibody fragments and/or full length BsAbs which enable the BsAbs to be expressed and recovered directly and/or efficiently from recombinant cell culture and/or which may be produced with efficient yields and purities at commercially reasonable costs.